Streptococcus pneumoniae is a major cause of invasive diseases such as meningitis, septicaemia and pneumonia. Approximately, one million children under 5 years of age die of pneumococcal disease annually (Jaffar S., et al., Vaccine, 1999; 18(7-8):633-40).
In countries where the incidence of Neisseria meningitidis and Haemophilus influenzae infections has drastically decreased through the introduction of vaccines against meningococci group C and H. influenzae type B, S. pneumoniae has become the major cause of meningitis and septicemia in children. In addition, the morbidity by S. pneumoniae through respiratory tract infections such as otitis media and sinusitis is enormous. Thirty to 50% of all patients with otitis media and a substantial percentage of cases of sinusitis and pneumonia are caused by pneumococci. Risk groups for serious pneumococcal disease include children under the age of 2 years, elderly and patients with immunodeficiencies (Pichichero M. E., et al., Pediatr. Infect. Dis. J., 1997; 16(1):72-4).
Nasopharyngeal colonization by S. pneumoniae is common: probably all humans are colonized with this organism at least once early in life. Especially in circumstances of crowding, as in day-care centers, nursing homes, hospitals and jails, the risk of colonization with pneumococci is high (Kellner J. D., et al., Arch. Pediatr. Adolesc. Med., 1999; 153(5):495-502; Nuorti J. P., et al., N. Engl. J. Med., 1998; 338(26):1861-8; Principi N., et al., Pediatr. Infect. Dis. J., 1999; 18(6):517-23).
Colonization is not usually followed by disease, since this is prevented by the innate and adaptive immune system. However, disturbance of homeostasis between host and pathogen, for example through viral infections, malnutrition or local damage of the mucosa, is associated with the development of invasive diseases (Hament J. M., et al., FEMS Immunol, Med, Microbiol. 1999; 26(3/4):189-95; Mulholland K., Vaccine, 1999; 17(Suppl 1):S79-84; Plotkowski M. C., et al., Am. Rev. Respir. Dis., 1986; 134(5):1040-4).
Despite the availability of various potential control measures, bacterial infections persist as major causes of morbidity and mortality. For example, despite the long-standing use of the 23-valent pneumococcal vaccine in specific at-risk group over the age of 2 years, the pneumococcus (Streptococcus pneumoniae) remains the leading cause of community-acquired pneumonia, otitis media and meningitis (Fedson D. S., Vaccine, 1999; 30; 17 Suppl. 1:S85-90. Review; Tuomanen E. I., Vaccine, 2000; 8; 19 Suppl. 1:S38-40. Review).
The recent introduction of the 7-valent conjugate pneumococcal vaccine into the childhood immunization schedules of some countries will likely reduce pneumococcal disease. However, this vaccine covers infections caused by only some pneumococcal serotypes and the replacement over time of these serotypes by resistant ones is a likely possibility. Problems exist also with therapeutic interventions, since many invasive S. pneumoniae strains are resistant to beta-lactam and other antibiotics (Neuman M. I., et al., J. Emerg. Med., 2007; 32(4):349-57).
In the last years, many pneumococcal proteins, including pneumolysin, surface protein A (PspA), surface adhesin A (PsaA), surface protein C (PspC), neuraminidase and autolysin, have been proposed as potential vaccine candidates (Briles D. E., et al., Vaccine, 2000; 8; 19 Suppl. 1:S87-95. Review).
In addition, intensive research is aimed towards discovery of novel targets for antibiotic treatment to overcome drug resistance (Bogaert D., et al., Vaccine, 2004; Sep. 28; 22(29-30):4014-20).
In the past years, one of the inventors and colleagues applied the technology of phage display to the identification of antigens of Toxoplasma gondii (Beghetto E., et al., Int. J. Parasitol., 2001; 31(14):1659-68; Beghetto E., et al., Int. J. Parasitol., 2003; 33(2):163-73; WO03/080839) and tumors (WO03/010199; WO03/011903).
In 2006 the present inventors focused their attention on the identification of pneumococcal proteins by using the technology of phage display.
The library screening allowed the isolation of phage clones carrying three distinct antigenic regions of a hypothetical pneumococcal protein, encoded by the open reading frame (ORF) spr0075 in the S. pneumoniae R6 strain genome sequence. This was the first identified S. pneumoniae gene product, having an antigenic function during infection (Beghetto et al., FEMS Microbial Lett., 2006; 262:14-21).
The spr0075 ORF in the R6 genome of S. pneumoniae encodes a putative protein of 1161 aa (GenBank accession no. NP357669), having an expected molecular mass of 123 kDa. Analysis of the Spr0075 protein sequence reveals the presence of: (a) a putative signal peptide, located between amino acids 1 and 40 (putative cleavage site aa 41), (b) six adjacent repeated regions (152 aa long) and (c) an LPxTG anchoring motif (Schneewind O Mihaylova-Petkov D & Model P 1993), in the C-terminal region (residues 1148-1152 aa). The spr0075 gene from the R6 strains is well preserved among several strains (type 19F, 6B, 2, 4, 23F), although the number of repeated regions may vary.
The protein Spr0075 is encoded by an spr0075 ORF in the R6 genome sequence (Hoskins J., et al., J. Bacteriol., 2001; 183:5709-5717) located between nucleotides 80186 and 83671.
Antigenic regions of Spr0075 protein reacted with more than 60% of sera, indicating a broad recognition of this protein antigen.
The analysis of virulence was conducted comparing FP242, the isogenic encapsulated mutant strain wherein spr0075 was deleted, with the wild type D 39.
Female CD mice, 5-8 week old were intravenously injected with 100 μl of PBS containing 70,000 CFU of D39 and the percentage of survival was followed up to 10 days. The results in FIG. 1 show that the virulence of the spr0075 deletion mutant was comparable to the wild type.
The efficacy of antipneumococcus capsular polysaccharide-based vaccines has been extensively debated, as the protection elicited by capsule polysaccharides is stringently serotype-specific (Hausdorff et al., 2005) and often unable to induce long-term memory response.
In the last generation of vaccines (Prevnar/Prevenar, 7-valent pneumococcal conjugate vaccine), purified capsular polysaccharides of seven S. pneumoniae strains were coupled with a protein carrier, in order to exceed the above limitations. The vaccine is effective in 97% of invasive diseases caused by vaccine serotypes and offers some protection against otitis media and pneumococcal carriage (Bogaert et al., 2004b).
Owing to the limited serotype coverage, risks of serotype replacement and the high cost of pneumococcal glycoconjugated vaccines, great interest in the development of formulations based on pneumococcal protein antigens has emerged in the last decade (Bogaert et al., 2004b).
It is strongly felt the need of vaccine compositions based on new antigen fragments, capable to recognize several serotypes and induce immune response to species with high variability or to different bacterial types.
In view of the prior art and starting from the results obtained on spr0075, the present inventors deeply investigated the genoma of pneumococcus by using the phage display technique, in order to find new antigens with the desired properties.
Surprisingly the inventors identified the sequences defined as spr1370, spr1875 and spr1120.
Said sequences are virulence factors isolated from Streptococcus pneumoniae and conserved in other bacteria. A virulence factor is a protein indispensable for bacteria propagation in the host.
Object of the present invention are antigen fragments and/or fragments containing an epitope with the following amino acid sequence:
SEQ SPM4(SEQ ID NO: 1)FISQAVAKYPTLLESLPVKDSGARYRLEGYLFPATYSIKESTTIESLIDEMLAAMDKNLSLYYSTIKSKNLTVNELLTIASLVEKEGAKTEDRKLIAGVFYNRLNRDMPLQSNIAILYAQGKLGQNISLAEDVAIDTNIDSPYNVYKNVGLMPGPVDSPSLDAIESSINQTKSDNLYFVADVTEGKVYYANNQEDHDRN SEQ Spr1370(SEQ ID NO: 2)MSEKSREEEKLSFKEQILRDLEKVKGYDEVLKEDEAVVRTPANEPSAEELMADSLSTVEEIMRKAPTVPTHPSQGVPASPADEIQRETPGVPSHPSQDVPSSPAEESGSRPGPGPVRPKKLEREYNETPTRVAVSYTTAEKKAEQAGPETPTPATETVDIIRDTSRRSRREGAKPAKPKKEKKSHVKAFVISFLVFLALLSAGGYFGYQYVLDSLLPIDANSKKYVTVGIPEGSNVQEIGTTLEKAGLVKHGLIFSFYAKYKNYTDLKAGYYNLQKSMSTEDLLKELQKGGTDEPQEPVLATLTIPEGYTLDQIAQTVGQLQGDFKESLTAEAFLAKVQDETFISQAVAKYPTLLESLPVKDSGARYRLEGYLFPATYSIKESTTIESLIDEMLAAMDKNLSLYYSTIKSKNLTVNELLTIASLVEKEGAKTEDRKLIAGVFYNRLNRDMPLQSNIAILYAQGKLGQNISLAEDVAIDTNIDSPYNVYKNVGLMPGPVDSPSLDAIESSINQTKSDNLYFVADVTEGKVYYANNQEDHDRNVAEHVNSKLN SEQ SPM8(SEQ ID NO: 3)GVKESSNIASYEDLKGKTVGVKNGTASQTFLTENQSKYGYKIKTFADGSSMDDSLNTGAIDAVMDDEPVLKYSISQGQKLKTPISGTPIGETAFAVKKGANPELIEMF SEQ Spr1120(SEQ ID NO: 4)MKKKFLAFLLILFPIFSLGIAKAETIKIVSDTAYAPFEFKDSDQTYKGIDVDIINKVAEIKGWNIQMSYPGFDAAVNAVQAGQADAIMAGMTKTKEREKVFTMSDTYYDTKVVIATTKSHKISKYDQLTGKTVGVKNGTAAQRFLETIKDKYGFTIKTFDTGDLMNNSLSAGAIDAMMDDKPVIEYAINQGQDLHIEMDGEAVGSFAFGVKKGSKYEHLVTEFNQALSEMKKDGSLDKIIKKWTASSSSAVPTTTTLAGLKAIPVKAKYIIASDSSFAPFVFQNSSNQYTGIDMELIKAIAKDQGFEIEITNPGFDAAISAVQAGQADGIIAGMSVTDARKATFDFSESYYTANTILGVKESSNIASYEDLKGKTVGVKNGTASQTFLTENQSKYGYKIKTFADGSSMDDSLNTGAIDAVMDDEPVLKYSISQGQKLKTPISGTPIGETAFAVKKGANPELIEMFNNGLANLKANGEFQKILDKYLASESSTASTSTVDETTLWGLLQNNYKQLLSGLGITLALALISFAIAIVIGIIFGMFSVSPYKSLRVISEIFVDVIRGIPLMILAAFIFWGIPNFIESITGQQSPINDFVAGTIALSLNAAAYIAEIVRGGIQAVPVGQMEASRSLGISYGKTMRKIILPQVTKLMLPNFVNQFVIALKDTTIVSAIGLVELFQTGKIIIARNYQSFKMYAILAIFYLVIITLLTRLAKRLEKRIR SEQ R4(SEQ ID NO: 5)EQIQNDLTKTDNKTSYTVQYGDTLSTIAEALGVDVTVLANLNKITNMDLIFPETVLTTTVNEAEEVTEVEIQTPQADSSEEVTTATADLTTNQVTVDDQTVQVADLSQPIAEAPKEVASSSEVTKTVIASEEVAPSTGTSVPEEQTAETSSAVAEEAPQET SEQ Spr1875(SEQ ID NO: 6)MKKRMLLASTVALSFAPVLATQAEEVLWTARSVEQIQNDLTKTDNKTSYTVQYGDTLSTIAEALGVDVTVLANLNKITNMDLIFPETVLTTTVNEAEEVTEVEIQTPQADSSEEVTTATADLTTNQVTVDDQTVQVADLSQPIAEAPKEVASSSEVTKTVIASEEVAPSTGTSVPEEQTAETSSAVAEEAPQETTPAEKQETQTSPQAASAVEATTTSSEAKEVASSNGATAAVSTYQPEETKIISTTYEAPAAPDYAGLAVAKSENAGLQPQTAAFKEEIANLFGITSFSGYRPGDSGDHGKGLAIDFMVPERSELGDKIAEYAIQNMASRGISYIIWKQRFYAPFDSKYGPANTWNPMPDRGSVTENHYDHVHVSMNG(wherein SEQ SPM4, SEQ SPM8 and SEQ R4 are the amino acid sequences of the fragments identified by using the technology of phage display while SEQ Spr1370, SEQ Spr1120 and SEQ Spr1875 are the amino acid sequences of the corresponding Open Reading Frame)and the corresponding coding nucleotide sequence:
SEQ SPM4(SEQ ID NO: 7)TTTATCAGTCAAGCAGTAGCGAAATATCCTACTTTACTGGAAAGTTTGCCTGTAAAAGACAGCGGTGCGCGTTATCGTTTGGAAGGATACCTTTTCCCAGCTACATACTCTATCAAGGAAAGCACAACTATTGAGAGCTTGATTGATGAGATGTTAGCTGCTATGGATAAGAACCTATCTCTTTACTATAGTACTATCAAATCTAAAAACTTGACTGTCAATGAGTTGTTGACCATTGCTTCCTTGGTCGAAAAAGAAGGTGCCAAGACAGAAGATCGTAAGCTCATTGCAGGTGTATTCTACAATCGTTTGAATCGTGATATGCCACTTCAAAGTAATATTGCAATCTTGTATGCCCAAGGAAAACTGGGGCAAAATATCAGTCTAGCTGAGGATGTTGCGATTGATACCAACATTGATTCACCTTATAATGTTTATAAAAATGTAGGTCTCATGCCTGGTCCAGTCGATAGTCCAAGTCTGGATGCGATTGAGTCAAGCATCAATCAAACTAAGAGCGATAACCTCTACTTTGTAGCAGATGTCACAGAAGGCAAGGTCTACTATGCTAACAATCAAGAAGACCACGACCGCA SEQ SPM8(SEQ ID NO: 8)GGTGTCAAAGAATCAAGTAATATTGCTTCTTATGAAGATCTAAAAGGAAAGACAGTCGGTGTTAAAAACGGAACTGCTTCTCAAACCTTCCTAACAGAAAATCAAAGCAAATACGGCTACAAAATCAAAACCTTTGCTGATGGTTCTTCAATGGATGACAGTTTAAACACTGGTGCCATTGATGCCGTTATGGATGATGAACCTGTTCTCAAATATTCTATCAGCCAAGGTCAAAAATTGAAAACTCCAATCTCTGGAACTCCAATCGGTGAAACAGCCTTTGCCGTTAAAAAAGGAGCAAATCCAGAACTGATTGAAATGTTC SEQ R4(SEQ ID NO: 9)GAGCAAATCCAAAACGATTTGACTAAAACGGACAACAAAACAAGTTATACCGTACAGTATGGTGATACTTTGAGCACCATTGCAGAAGCCTTGGGTGTAGATGTCACAGTGCTTGCGAATCTGAACAAAATCACTAATATGGACTTGATTTTCCCAGAAACTGTTTTGACAACGACTGTCAATGAAGCAGAAGAAGTAACAGAAGTTGAAATCCAAACACCTCAAGCAGACTCTAGTGAAGAAGTGACAACTGCGACAGCAGATTTGACCACTAATCAAGTGACCGTTGATGATCAAACTGTTCAGGTTGCAGACCTTTCTCAACCAATTGCAGAAGCTCCAAAAGAAGTAGCATCAAGTTCAGAAGTTACAAAGACAGTGATTGCTTCTGAAGAAGTGGCACCATCTACGGGCACTTCTGTCCCAGAGGAGCAAACGGCCGAAACAAGCAGTGCAGTTGCAGAAGAAGCTCCTCAGGAAACGand the hybridizing nucleotide sequences, also under stringent hybridization, thereof. In this contest the terms “hybridization” and “stringent” refer to the conventional hybridization techniques well known to the person skilled in this field (Buzdin A and Lukyanov S (eds) Nucleic Acids Hybridization Kluwer Academic Publishers Netherlands 2007).
Another object of the present invention is a method for the identification of the amino acid sequences above disclosed comprising the following steps:                a) obtaining a serum pool from subjects immunized with a killed bacterial strain;        b) administering to subjects the serum pool obtained in step a) to give immunized subjects;        c) collecting the sera from said immunized subjects obtained in step b), and        d) undergoing the sera of step c to phage display technique.)        
In the context of the above method according to the present invention, a “subject” is for example a laboratory animal, such as a mouse.
Another object of the present invention is a method for the identification of the above antigen fragments and/or fragments containing epitopes by means of selection of libraries of cDNA or DNA fragments of Streptococcus pneumoniae with sera of subjects immunized with the killed Streptococcus pneumoniae. 
A further object of the present invention is the use of said antigen fragments as active agents for the diagnosis of pneumococcal infections, in particular Streptococcus pneumoniae infections, Streptococcus gordonii infections, Streptococcus sanguinis infections, Streptococcus thermophilus infections, Streptococcus suis infections, Streptococcus agalactiae infections, Streptococcus pyogenes infections, Streptococcus mutans infections, Enterococcus faecalis infections, Enterococcus faecium infections, Rhodococcus sp. infections.
It is also object of the present invention, the use of said antigen fragments for the preparation of a medicament, preferably for the prevention or the treatment of pneumococcal infections, such as the ones listed above.
Are object of the present invention also the specific ligands such as natural host ligands (eg complement and other opsonins) or artificial ligands such as peptides selected with the above antigen using random peptide libraries and any molecules that bind to the above epitopes and the anti-epitope antibodies raised against said epitopes, and the use of at least one of said ligands and/or at least one of said antibodies for the preparation of means for the diagnosis of pneumococcal infections, such as the ones listed above.
Another object of the present invention is a method for the diagnosis of pneumococcal infections comprising the selection of sera of subjects affected by or suspected of being affected by said infection with the above antigen fragments and/or with at least one of the above ligands and/or at least one of the above antibodies and a diagnostic kit for pneumococcal infections.
A further object of the present invention is a pharmaceutical composition, particularly in the form of a vaccine, containing at least one of the above antigen fragments or one of the above sequences. Said composition is suitable for human and/or veterinary use.